Submitted July 28, 1997.
Immortalization of human cells in culture is usually associated with
expression of telomerase activity. In some cases, however, no
telomerase activity is detectable even though comparison of the
terminal restriction fragment (TRF) pattern before and after
immortalization shows that lengthening of telomeres has occurred. The
extreme heterogeneity in telomere length and the differences in the
dynamics of telomere maintenance in telomerase-negative cell lines
compared to telomerase-positive cell lines indicate that these cells
have utilized one or more alternative mechanisms for lengthening of
telomeres (ALT). All telomerase-negative immortalized cell lines
examined to date show evidence of ALT activity, consistent with the
hypothesis that telomere maintenance either by telomerase or by ALT is
required for immortalization. The nature of the ALT mechanism(s) is
currently unknown, but studies of telomere dynamics in an ALT cell line
containing a marker just proximal to the telomeric sequences show
gradual shortening of the telomere followed by rapid elongation. This
is consistent with a non-reciprocal recombinational mechanism similar
to that found in telomerase-defective mutant yeast strains.
KEY WORDS: telomeres, alternative lengthening of telomeres, ALT,
telomerase, senescence, crisis, terminal proliferation arrest.

According to the telomere hypothesis of senescence, a permanent cell
cycle arrest is triggered when one or more telomeres are shortened
beyond a critical length [1-3].
Consistent with this hypothesis, there is experimental evidence that
telomeres shorten when normal cells undergo replication [4]. The reasons for this telomere shortening are not
entirely clear, but may include the so-called "end-replication
problem" where removal of the most distal RNA primer is thought to
result in failure of the last few bases of the telomere to be
replicated [5], and the activity of a putative
exonuclease that removes the terminal 100-200 nucleotides of the C-rich
strand of telomeric DNA [6].

Whether the telomeres of senescent cells have truly shortened to a
critical length is unclear, however, because experiments in which
senescence is over-ridden by expression of DNA tumor virus oncoproteins
[7-9] or by loss of wild-type
p53 [10] have shown that telomeres can continue to
shorten when further cell division occurs. Although the loss of
function of genes involved in triggering senescence [11] can result in a temporary escape from senescence,
after further proliferation and telomere shortening the cells enter
another terminal proliferation arrest state known as crisis [12]. The increase in chromosome fusions and cell
death associated with crisis suggest that the shortened telomere length
of cells in crisis is incompatible with normal function.

Cells that escape from crisis are usually capable of unlimited
proliferation, i.e., they are immortalized. A prediction of the
telomere hypothesis of senescence is that immortalized cells must have
some process for preventing further telomere shortening. In most
immortalized cell lines, this process involves the activity of a
multi-subunit enzyme, telomerase [13]. Telomerase
uses an RNA template for synthesizing telomeric DNA repeats to replace
those that are lost as a result of cell division. In some cell lines,
however, no telomerase activity is detectable. In this article the
evidence that these cell lines have an alternative mechanism for the
lengthening of telomeres (ALT) will be reviewed.

In vitro Immortalized Cell Lines without Telomerase
Activity

Some human cell lines immortalized in vitro were found to have no
detectable telomerase activity (Fig. 1) [10, 14-17]
in the sensitive telomeric repeat amplification protocol (TRAP) assay
[15]. Mixing experiments yielded no evidence that
these cell lines contain inhibitors of telomerase itself or of the TRAP
assay [16]. Similarly, dilution of TRAP assay
samples did not reveal evidence of the presence of telomerase
inhibitors (Fig. 2).

Fig. 1. Telomerase activity before and after immortalization.
Lysates from the human cell lines indicated were subjected to TRAP
assay before crisis (pre) and after escape from crisis (post). The cell
lines were derived from fibroblasts (BFT-3K, BFT-3I, IIICF-T/A6, and
BFT-3B), mesothelial cells (MeT-5A), and bronchial epithelial cells
(HB56B/5T, BET-3M, BES-1A1, and BET-1A). LB is the lysis buffer
negative control; positive controls are lysates equivalent to 100 and
1000 HeLa cells. Telomerase activity is detected as a 6 base-pair
ladder following electrophoresis of radiolabeled reaction products on a
polyacrylamide gel. For each of the cell lines shown, no telomerase was
detected before immortalization. In the majority, telomerase was seen
after immortalization, but in some cells (IIICF-T/A6 and BET-3M) no
telomerase activity was detectable.

Fig. 2. Dilution of cell extracts did not reveal telomerase
activity in a telomerase-negative immortalized cell line, IIICF/c. For
cell lines IIICF/c and HeLa, the indicated amount of protein was
assayed by TRAP. LB is the lysis buffer negative control, and lysates
equivalent to 1000, 100, and 10 HeLa cells were used as the positive
controls. Samples with 10 µg of HeLa extract had less TRAP
activity than the lower concentrations indicating the presence of a
dilutable inhibitor, but dilution of the IIICF/c extract did not reveal
any evidence of telomerase activity.

Although most of the cell lines studied are SV40-immortalized
fibroblasts, lines derived from other cell types and immortalized in
other ways (including the use of other DNA tumor virus genes, chemical
carcinogens, and spontaneously in the case of fibroblasts from a
Li--Fraumeni syndrome (LFS) family member) can also be
telomerase-negative [16]. Although the number of
epithelial and mesothelial cell lines studied so far is small, they
seem more likely to be telomerase-positive than cell lines derived from
fibroblasts [18].

Overall, approximately one-quarter of all in vitro immortalized
cell lines are telomerase-negative [18]. Some
fibroblast strains, however, seem to have a propensity for
immortalization in the absence of telomerase activity. Each of 10 cell
lines derived from breast fibroblasts of an LFS individual by
spontaneous immortalization or following transfection with SV40 or
human papilloma virus HPV16 genes was telomerase-negative [16]. Eleven of 20 cell lines derived by SV40 gene
transfection of jejunal fibroblasts from a non-LFS individual were
telomerase-negative (P. Bonnefin et al., unpublished data). In one
study 19 of 19 SV40-immortalized neonatal foreskin fibroblasts were
telomerase-positive [19], but in another study 3
of 3 SV40-immortalized neonatal foreskin fibroblasts were
telomerase-negative (J. Murnane, unpublished data). The reasons for
these strain differences are not clear but could include p53 status
(the LFS cells are heterozygous wild-type/null-mutant for p53) or the
differentiation status of the cell strain.

Evidence for an Alternative Mechanism for Lengthening Telomeres
(ALT)

Several lines of evidence indicate that the telomeres of
telomerase-negative immortalized cell lines undergo lengthening.
Telomere length is usually assessed either by gel electrophoresis or by
chromosomal in situ hybridization. In the electrophoretic
approach, genomic DNA digested with restriction enzymes that cut
frequently throughout the genome but not within the telomeric repeat
sequence is size-fractionated by electrophoresis and then probed with a
labeled oligonucleotide (TTAGGG)n corresponding to
multiples of the telomeric repeat sequence. This method detects
terminal restriction fragments (TRFs) that consist of telomeric DNA
plus a small amount of subtelomeric DNA between the telomere and the
closest restriction site. Telomerase-negative immortalized cell lines
have a TRF pattern that is distinct from immortalized cell lines with
telomerase activity (Fig. 3): there is a smear from
the bottom of the gel up to the well indicating that the TRF sizes
range from very small to extremely large [16].
In situ hybridization studies using conventional [20] or peptide nucleic acid [21]
probes have shown that this heterogeneity of telomere length occurs
within individual telomerase-negative cells. The presence of the very
long telomeres indicates that telomere lengthening has occurred. This
characteristic pattern has been found in all telomerase-negative cell
lines examined by TRF analysis to date and has never been seen in any
telomerase-positive cell line where telomeres are generally much more
homogeneous in length.

Fig. 3. Immortalized cell lines lacking telomerase activity have
very long telomeres. Terminal restriction fragments (TRFs) were
electrophoresed and detected with a radiolabeled telomere repeat
sequence probe. Telomerase activity of the cell lines shown is
indicated by "tel". The telomerase-negative immortalized
cells all have very heterogeneous TRF lengths ranging from very short
to very long (on the left, in kb). For two telomerase-negative cell
lines, IIICF-T/A6 and BET-3M, TRF analysis is shown pre- and
post-immortalization. In both cases, telomere lengthening has occurred
following immortalization.

One possibility that has been considered is that the telomerase-negative
cells maintain their telomeres by intermittent telomerase activity. If
true, their telomeres would be expected to undergo shortening in the
periods when telomerase is absent. However, no shortening was detected
in one cell line, GM847, that was analyzed over the course of 98
population doublings (PD) [16]. In two other cell
lines, IIICF-T/A6 and IIICF/c, there was evidence for an increase in
the amount of large TRFs following immortalization and no subsequent
decrease in mean TRF size despite the continued absence of detectable
telomerase for 95 PD [16] and >600 PD ([10] and T. Bryan et al., unpublished data),
respectively. Analysis of several additional telomerase-negative
cultures before and after immortalization (Fig. 3)
showed that in each case an increase in mean TRF size had occurred [16]. Since most of these cultures were clonally
derived it is clear that telomere elongation had occurred, rather than
the selection of a pre-existing clone with long telomeres.

These data show that telomere elongation occurs in the absence of
telomerase activity and thus indicate the existence of one or more
alternative mechanisms for lengthening telomeres (ALT). Even though all
of the ALT cell lines examined to date have a similar TRF pattern, it
cannot be assumed at this stage that the mechanism of telomere
lengthening is the same in each case.

In one telomerase-negative cell line, KB319, it has been possible to
follow the dynamics of changes in the length of a single telomere [14]. This cell line has a plasmid inserted proximal
to the repeat sequences of the telomere of chromosome 13q, so that
probing of TRF gels with labeled plasmid detects the TRF pattern of
only the marked chromosome. Three subclones of this cell line were
studied: one with a long telomere on chromosome 13, one with a short
telomere, and one in which no distinct band was discernible. These
subclones were subcloned further and the TRF analyses showed the
occurrence of both gradual and rapid length changes that sometimes
involved many kilobases. The gradual changes were due to the shortening
of telomeres at a rate consistent with that reported for telomeres of
telomerase-negative normal somatic cells, eventually resulting in the
loss of nearly all of the telomere. However, polymorphism was never
detected in the subtelomeric plasmid sequences and no increase in the
frequency of telomere associations occurred, indicating that the
telomeres were not completely lost. Instead, the short telomeres were
selectively elongated, indicating that a mechanism to recognize and
salvage overly short telomeres exists in this cell line. The
alternative possibility, that cells with complete telomere erosion were
lost from the population seems unlikely because there was no indication
of increased cell death or changes in cell doubling time during this
process. The marked telomere also showed rapid, highly heterogeneous
increases and decreases in length [14]. The
frequency of these rapid changes in length varied in different
subclones and showed a correlation with increased telomere
associations, suggesting that the complete loss of telomeres could
occur by these rapid events.

Despite different initial lengths, as a result of the dramatic increases
and decreases in length, in different KB319 subclones the telomere
marked by the plasmid insertion eventually took on the heterogeneous
distribution in length characteristic of telomeres in cells utilizing
ALT. The analysis of the dynamics of changes in the length of an
individual telomere therefore demonstrates that telomerase-negative
SV40-immortalized human cell lines continually maintain telomere
length.

ALT in Tumor Cell Lines and Tumors

ALT is not restricted to in vitro immortalized cell lines, but
has been found in 4 of 56 tumor-derived cell lines and 4 of 57 tumor
samples (T. Bryan et al., unpublished data). Interestingly, 3 of the 4
ALT cell lines were derived from sarcomas. This is consistent with the
observation that immortalized cell lines derived in vitro from
fibroblasts are more commonly ALT-positive than lines derived from
epithelial cells [18]. Since 90% of human tumors
are of epithelial origin, this may account in part for the low
incidence of ALT-positive tumors. There also appeared to be a possible
association between ALT and tumors arising in Li--Fraumeni syndrome
individuals (T. Bryan et al., unpublished data). The reasons for this
putative association are not known since the majority of human tumors
also contain p53 mutations but have telomerase activity. The first
genetic "event" in the process of oncogenesis is the
inherited p53 mutation in these cells, and maybe the order in which
mutations occur is important in determining the probability of
immortalization via the ALT pathway. The results of other studies are
also consistent with the presence of ALT in tumors. For example, 2 of
56 renal cell carcinomas were telomerase-negative and contained cell
clones with long TRFs [22].

A number of telomerase-negative tumors have been found to have telomeres
that are much shorter than those characteristic of ALT (T. Bryan et
al., unpublished data). It is therefore possible that some of these
tumors do not contain immortalized cells or that there may be
non-telomerase mechanisms of telomere maintenance that do not result in
very long telomeres. Conversely, a minority of telomerase-positive
tumors have very long telomeres indistinguishable from those of ALT. It
is possible that mutations in a telomerase component or in a
telomere-binding protein result in "runaway elongation" of
telomeres by telomerase, as has been observed in Tetrahymena and
the yeast Kluyveromyces lactis [23, 24]. Alternatively, there may be mixtures of
ALT-positive and telomerase-positive cells in the same tumor, or ALT
and telomerase may co-exist within the same cells. In the absence of
direct assays for ALT, it is not presently possible to distinguish
among these possibilities.

Possible Mechanisms of ALT

In considering the possible nature of the ALT mechanism(s), it may be
instructive to note that in addition to telomerase there are two other
mechanisms used by eukaryotes for maintenance of telomere length:
recombination and retrotransposition. Wang and Zakian transferred
telomere sequences from ciliates into the yeast, S. cerevisiae
and demonstrated recombination of these sequences [25]. Consequently, they proposed that
telomere--telomere recombination provides an efficient mechanism for
rescue of DNA termini with very short stretches of telomeric DNA. S.
cerevisiae strains lacking a functional EST1 gene underwent
progressive telomere shortening and growth senescence, but a minor
subpopulation was able to survive as the result of amplification and
acquisition of subtelomeric elements [26]. This
survival mechanism was shown to be dependent on RAD52, a protein
involved in homologous recombination and double-strand break repair in
a wide range of eukaryotes [27]. It was initially
thought that this process might be restricted to S. cerevisiae
because of the presence in this organism of large subtelomeric Y´
elements interspersed with telomeric repeats. However, deletion of the
telomerase RNA gene from the yeast K. lactis, which does not
contain subtelomeric telomere-like repeats also yielded survivors at a
relatively high frequency. In this instance recombination of the
telomere repeat sequences themselves was involved, which was also
dependent on RAD52 [28]. The authors proposed that
in the presence of telomerase activity the normal-length telomeres are
capped with proteins that prevent recombination events, but that
severely shortened telomeres become uncapped, promoting recombinational
repair [28]. A similar mechanism was previously
proposed to explain the rapid elongation of short telomeres in a
telomerase-negative human cell line [14]. Similar
mechanisms may be involved in telomere maintenance in
telomerase-negative yeast and human cells, because the TRF pattern of
the est1 S. cerevisiae and the telomerase-negative K.
lactis resembled those seen in human ALT cell lines.

Analysis of the KB319 line suggests that ALT cells are capable of
seeding new telomeres following chromosome breakage. Transfected
plasmid DNA integrated at the end of a chromosome had telomeric repeat
sequences added onto both ends, one end of which became the new
telomere of this chromosome [29]. Other studies
have also suggested that telomerase can mediate the de novo
addition of telomere sequence in the healing of broken chromosomes [30]. In support of the possibility that ALT may
involve recombination, it is of interest to note that addition of
telomeres to broken yeast chromosomes may be mediated either by
telomerase or by recombination [25, 31].

In Drosophila melanogaster and related dipterans non-LTR
retroposons of the TART and HeT-A classes are utilized instead of short
repeat sequences to replace the sequences lost from the end of
chromosomes during cell division [32-37]. The telomeres of Drosophila are capped by
chains of retroposons which undergo gradual shortening, and which, to
compensate this loss, can be lengthened by a retrotransposition event.
This mechanism is also able to heal broken chromosomes in
Drosophila. Although it is not known whether retrotransposition
can be utilized to lengthen telomeres in eukaryotes distant from
Drosophila, an abnormally shortened telomere may well be
perceived by the cell as a double-strand break and it has been shown
that retroelements can mediate the repair of double-strand breaks in
yeast in the absence of RAD52 [38, 39]. In yeast that are mutant at both the telomerase
RNA gene and the RAD52 loci there are rare survivors which occur
through a mechanism which is currently unknown [28]. Although it is possible that these survivors
have retained some residual recombination activity, either
RAD52-dependent or -independent, it will be interesting to determine
whether retrotransposition is involved in maintenance of their
telomeres.

The above data suggest that there are three telomere maintenance
mechanisms (telomerase, retrotransposition, and recombination) that are
conserved in most eukaryotes. Drosophila and related organisms
might therefore be considered to be exceptions in which one of the
three mechanisms, telomerase, has not been conserved and the
retrotransposition mechanism which may serve as a "backup" in
most other eukaryotes is preferentially used. However, the recent
finding that the active site of yeast and Euplotes aediculatus
telomerase is a reverse transcriptase that is closely related to the
Drosophila TART retrotransposon, may suggest that the
retrotransposition mechanism of telomere maintenance in
Drosophila can be regarded as somewhat similar to the use of
telomerase in other eukaryotes [40]. Thus, there
may be only two main categories of telomere maintenance
mechanisms--telomerase and recombination.

In view of the above considerations, both recombination and
retrotransposition should be considered as candidate mechanisms of ALT.
Detailed sequence data are not available for ALT telomeres at present,
but hybridization of TRFs with telomere repeat probes has shown that
they contain sequences that are closely related to the telomere repeat
and that the amount of this sequence increases in ALT cells [16]. Therefore, recombination is more likely to be
the mechanism of ALT than retrotransposition. A proposed model for
recombination-mediated elongation of telomeres is shown in Fig. 4. It is important to keep in mind, however, that
there may be more than one ALT mechanism.

Fig. 4. A model for recombination-mediated lengthening of
telomeres [25, 28]. a) When a
telomere becomes critically short it may be interpreted by the cell as
a double-strand break (DSB). b) The DSB repair enzymes then mediate
invasion by a single-stranded 3´ end of the short telomere
between the strands of a longer telomere. This step may be dependent on
RAD52. DNA polymerase may then extend the short strand, using the long
strand as the template. c) The crossed-over strands may then be subject
to cleavage by a nuclease (-->) followed by ligation, resulting in
recombinant DNA molecules (d), as has been proposed for DSB repair by
recombination in yeast [60]. Alternatively, the
structure shown in (b) may be resolved by unwinding of the newly formed
helix and rewinding (c), resulting in non-recombinant molecules (d).
This has been shown to occur in DSB repair in yeast [61]. In either case, the staggered annealing of
repeats in the short and long telomeres results in net telomere
elongation.

A possibility that seems unlikely but cannot be ruled out at present is
that ALT cells might contain an abnormal telomerase activity with the
following properties: 1) its regulation is abnormal, resulting in
telomere lengths ranging from short to very long; and 2) it is not able
to be detected by conventional or TRAP assays. Evidence is accumulating
for the role of telomere-binding proteins (TBPs) in feedback control of
telomere length [41, 42], and
certain mutations in the telomerase RNA gene (especially those that
result in an abnormal telomere sequence that affects binding of TBPs)
or mutations in TBPs themselves have been shown to cause "runaway
elongation" of telomeres in Tetrahymena and the yeast K.
lactis [23, 24, 43-46]. The combination of no
detectable telomerase with heterogeneous and abnormally long telomeres
could theoretically be produced if such mutations in a telomerase
component or a TBP resulted both in dysregulated telomere elongation
activity in vivo and loss of activity in the available in
vitro assays. Alterations in TBPs were proposed as a possible
mechanism for the rapid changes in the length of an individual telomere
in a telomerase-negative cell line [14]. However,
at present there is no evidence for changes in TBPs in cell lines
expressing ALT, and indeed in some ALT cell lines there is no
detectable expression of the human telomerase RNA (hTR) gene, making it
very unlikely that telomerase is involved, at least in those cells [47]. Furthermore, in ALT cell lines that express hTR,
no hTR mutations likely to cause dysregulated lengthening were found
[47].

Do Normal Cells Repress ALT?

Given the postulated importance of telomere maintenance to
immortalization, and the well-documented ability of normal cells to
suppress the immortal phenotype in normal--immortal hybrids, it seems
likely that normal somatic cells contain repressors of ALT. The
available data are consistent with this prediction, but definitive
evidence is currently lacking. For example, a cell line GM847 that was
subsequently found to be ALT-positive [16]
exhibited a finite lifespan when fused with immortalized cell lines
from other complementation groups [48-50], implying that telomere maintenance may have been
repressed in the hybrids. When GM847 was fused with telomerase-positive
immortalized cell lines, the senescent hybrid progeny were mostly
telomerase-negative but there were insufficient cells to obtain DNA to
determine whether the TRF pattern typical of ALT had also disappeared
[16]. A large number of genes encoding helicases,
topoisomerases and polymerases, have been identified that repress
homologous recombination in yeast [51]. It is
possible that the mammalian homologs of one or more of these genes may
be responsible for repressing ALT in normal mammalian cells. In view of
the apparent predilection for ALT in LFS cells, p53 must be considered
as a potential contributor to the repression of ALT in normal cells. A
possible mechanism may be suggested by the observation that both RAD52
and p53 bind RAD51 [52, 53].

We have addressed the question of whether the protein product of the
ataxia-telangiectasia (AT) gene, ATM, might be involved in telomere
maintenance. ATM has a relatively high degree of homology to the yeast
TEL1 gene [54] which has been demonstrated
to influence telomere length [55]. AT cells are
reported to exhibit a high rate of end-to-end chromosomal fusions [56] and lymphocytes from AT individuals undergo
accelerated telomere shortening [57], suggesting
the possibility that ATM may normally be involved in telomere
stability. Further, AT cells display elevated levels of
intrachromosomal recombination [58]. Of 8
SV40-immortalized AT fibroblast lines, 5 were found to be
telomerase-positive and 3 were ALT-positive indicating either that an
SV40 gene product is capable of substituting for some aspect of ATM
function or that ATM is not required for telomere maintenance via
telomerase or ALT [59].

Clinical Implications of ALT

Regardless of the precise mechanism(s) involved, the existence of ALT
has obvious implications for proposals to treat cancer with inhibitors
of telomerase. Although ALT appears to be present in only a minority of
tumors, effective inhibition of telomerase activity will subject tumors
to strong selection pressure for the emergence of treatment-resistant
cells via activation of ALT. A detailed understanding of ALT may make
treatment with a combination of inhibitors of all telomere maintenance
mechanisms feasible.

Work in the authors' laboratories was supported by the Carcinogenesis
Fellowship of the New South Wales Cancer Council (to R. Reddel), a
National Health and Medical Research Council of Australia postgraduate
scholarship (to T. Bryan), and grant No. 3RO1CA69044-01S1 from the
National Cancer Institute, NIH, USA. We thank Lindy Hodgkin for
assistance in preparing the manuscript.